Transcription factors play important roles in regulation of gene expression and phenotype. A variety of approaches have been utilized to develop gene regulatory networks to predict the regulatory targets for each transcription factor, such as yeast-1-hybrid screens and gene co-expression network analysis. Here we identified potential transcription factor targets and used a reverse genetics approach to test the predictions of several gene regulatory networks in maize. Loss-of-function mutant alleles were isolated for 22 maize transcription factors. These mutants did not exhibit obvious morphological phenotypes. However, transcriptomic profiling identified differentially expressed genes in each of the mutant genotypes, and targeted metabolic profiling indicated variable phenolic accumulation in some mutants. An analysis of expression levels for predicted target genes based on yeast-1-hybrid screens identified a small subset of predicted targets that exhibit altered expression levels. The analysis of predicted targets from gene co-expression network-based methods found significant enrichments for prediction sets of some transcription factors, but most predicted targets did not exhibit altered expression. This could result from false-positive gene co-expression network predictions, a transcription factor with a secondary regulatory role resulting in minor effects on gene regulation, or redundant gene regulation by other transcription factors. Collectively, these findings suggest that loss-of-function for single uncharacterized transcription factors might have limited phenotypic impacts but can reveal subsets of gene regulatory network predicted targets with altered expression.

Centrioles are subcellular organelles found at the cilia base with an evolutionarily conserved structure and a shock absorber-like function. In sperm, centrioles are found at the flagellum base and are essential for embryo development in basal animals. Yet, sperm centrioles have evolved diverse forms, sometimes acting like a transmission system, as in cattle, and sometimes becoming dispensable, as in house mice. How the essential sperm centriole evolved to become dispensable in some organisms is unclear. Here, we test the hypothesis that this transition occurred through a cascade of evolutionary changes to the proteins, structure, and function of sperm centrioles and was possibly driven by sperm competition. We found that the final steps in this cascade are associated with a change in the primary structure of the centriolar inner scaffold protein FAM161A in rodents. This information provides the first insight into the molecular mechanisms and adaptive evolution underlying a major evolutionary transition within the internal structure of the mammalian sperm neck.

Grass species, which include the major cereal crops maize, wheat, rice, and sugarcane, are an integral part of our global agriculture and source of food and energy for a growing world population. GRASSIUS was established as a knowledgebase for transcription factors (TFs) and coregulators (CoRegs) in maize and several other species in the grass family. TFs are a primary component of the gene regulatory networks (GRNs) and the underlying gene regulatory grids (GRGs) that govern all aspects of plant growth and metabolism. GRASSIUS is the source for all information pertaining to the maize TFome collection, which serves as a powerful resource for the discovery of GRNs in maize and other cereals [1,2]. Here we describe the release of the GRASSIUS 2.0 knowledgebase (www. grassius.org) with updated data, query, and tool features, as well as the ability to expand to accommodate future datasets. The membership and annotation of all TF and CoReg families has been updated and revised to include gene models from v3, v4, and v5, of the maize B73 genome and recent genome versions of rice, sorghum, Brachypodium, and sugarcane. A translation tool enables cross referencing of Gene IDs between versions of the maize genome. Protein-DNA interactions (PDIs) have been added incorporating results derived from various gene- and TF-centered PDI discovery tools and visualized through a new web-based interface. A filtering tools permits the selection and visualization of PDIs within a +/- 2 kb distance from the transcriptional start site (TSS) of a gene of interest. A new BLAST (Basic Local Alignment Search Tool) tool facilitates searching of the maize TFome as well as v3, v4, and v5 TF gene model sets. Lastly, we describe the methodology used to implement GRASSIUS 2.0 which can guide others in developing and updating similar plant gene regulatory knowledgebases.

There is strong interest in deciphering the gene regulatory networks (GRNs) that govern plant specialized metabolism to assist in plant breeding. Here, we investigated the GRN governing phenolic biosynthesis pathways from which similar to 8000 secondary metabolites are derived in plants. Previously it was established that 19 predominantly expressed phenolic (PEP) genes in maize are sufficient to explain >70 % of the metabolic flux through the core phenylpropanoid, monolignol, and flavonoid branches of this pathway. A yeast-1-hybrid (Y1H) gene centric screening approach was employed to discover upper level (tier 2, 3, and 4) regulators of maize PEP genes. These regulators were further examined by co-expression analyses, and a subset of protein-DNA interactions (PDIs) validated in vivo by ChIP-qPCR and luciferase reporter assays in maize protoplasts. This study reveals a comprehensive GRN composed of 429 PDIs that exhibits hubs with high connectivity and cross hierarchical regulation of PEP genes in different branches of the pathway. The core GRN includes TFs that are conserved in other plant species and that are implicated in phenolic gene regulation including ZmMYB40/53/100, ZmMADS9, and ZmWD40.1/PAC1. The GRN also includes conserved TFs (e.g., ZmC3H9, ZmHB20/79, ZmNAC103/123, ZmMYB19/26, ZmMYBR87, ZmDOF3, ZmbZIP67, ZmTCP30, and ZmbHLH128) which indicate that maize PEP genes are developmentally regulated but also fall under the control of biotic and abiotic stress signals. Together, the maize PEP GRN provides a complex regulatory mechanism that has evolved to coordinately regulate many phenolic genes in response to multiple internal and external signals and can guide efforts aimed at manipulating phenolic levels in plants towards targeted breeding improvement.

The amount of biological data is growing at a rapid pace as many high-throughput omics technologies and data pipelines are developed. This is resulting in the growth of databases for DNA and protein sequences, gene expression, protein accumulation, structural, and localization information. The diversity and multi-omics nature of such bioinformatic data requires well-designed databases for flexible organization and presentation. Besides general-purpose online bioinformatic databases, users need narrowly focused online databases to quickly access a meaningful collection of related data for their research. Here, we describe the methodology used to implement a plant gene regulatory knowledgebase, with data, query, and tool features, as well as the ability to expand to accommodate future datasets. We exemplify this methodology for the GRASSIUS knowledgebase, but it is applicable to developing and updating similar plant gene regulatory knowledgebases. GRASSIUS organizes and presents gene regulatory data from grass species with a central focus on maize (Zea mays). The main class of data presented include not only the families of transcription factors (TFs) and co-regulators (CRs) but also protein–DNA interaction data, where available.

Chloroplasts need to import preproteins and amino acids from the cytosol during their light-induced differentiation. Similarly, chloroplasts have to export organic matter including proteins and amino acids during leaf senescence. Members of the PRAT (preprotein and amino acid transporter) family are candidate transporters for both processes. Here, we defined the role of two small PRAT gene families, At4g26670 and At5g55510 (HP20 subfamily) versus At3g49560 and At5g24650 (HP30 subfamily) during greening of etiolated plants and during leaf senescence. Using a combination of reverse genetics, protein biochemistry and physiological tools, evidence was obtained for a role of chloroplast HP20, HP30 and HP30-2 in protein, but not amino acid, import into chloroplasts. HP20, HP30 and HP30-2 form larger complexes involved in the uptake of transit sequence-less cytosolic precursors. In addition, we identified a fraction of HP30-2 in mitochondria where it served a similar function as found for chloroplasts and operated in the uptake of transit sequence-less cytosolic precursor proteins. By contrast, HP22 was found to act in the export of proteins from chloroplasts during leaf senescence, and thus its role is entirely different from that of its orthologue, HP20. HP22 is part of a unique protein complex in the envelope of senescing chloroplasts that comprises at least 11 proteins and contains with HP65b (At5g55220) a protein that is related to the bacterial trigger factor chaperone. An ortholog of HP65b exists in the cyanobacterium Synechocystis and has previously been implicated in protein secretion. Whereas plants depleted of either HP22 or HP65b or even both were increasingly delayed in leaf senescence and retained much longer stromal chloroplast constituents than wild-type plants, HP22 overexpressors showed premature leaf senescence that was associated with accelerated losses of stromal chloroplast proteins. Together, our results identify the PRAT protein family as a unique system for importing and exporting proteins from chloroplasts.

Aminoacyl-tRNA synthetases (AaRS) charge tRNAs with amino acids for protein translation. In plants, cytoplasmic, mitochondrial, and chloroplast AaRS exist that are all coded for by nuclear genes and must be imported from the cytosol. In addition, only a few of the mitochondrial tRNAs needed for translation are encoded in mitochondrial DNA. Despite considerable progress made over the last few years, still little is known how the bulk of cytosolic AaRS and respective tRNAs are transported into mitochondria. Here, we report the identification of a protein complex that ties AaRS and tRNA import into the mitochondria of Arabidopsis thaliana. Using leucyl-tRNA synthetase 2 (LeuRS2) as a model for a mitochondrial signal peptide (MSP)-less precursor, a ≈30 kDa protein was identified that interacts with LeuRS2 during import. The protein identified is identical with a previously characterized mitochondrial protein designated HP30-2 (encoded by At3g49560) that contains a sterile alpha motif (SAM) similar to that found in RNA binding proteins. HP30-2 is part of a larger protein complex that contains with TIM22, TIM8, TIM9 and TIM10 four previously identified components of the translocase for MSP-less precursors. Lack of HP30-2 perturbed mitochondrial biogenesis and function and caused seedling lethality during greening, suggesting an essential role of HP30-2 in planta.

Higher plants contain a small, 5-member family of Rieske non-heme oxygenases that comprise the inner plastid envelope protein TIC55, phaeophorbide a oxygenasee (PAO), chlorophyllide a oxygenase (CAO), choline monooxygenase, and a 52 kDa protein (PTC52) associated with the precursor NADPH:protochlorophyllide (Pchlide) oxidoreductase A (pPORA) A translocon (PTC). Some of these chloroplast proteins have documented roles in chlorophyll biosynthesis (CAO) and degradation (PAO and TIC55), whereas the function of PTC52 remains unresolved. Biochemical evidence provided here identifies PTC52 as Pchlide a oxygenase of the inner plastid envelope linking Pchlide b synthesis to pPORA import. Protochlorophyllide b is the preferred substrate of PORA and its lack no longer allows pPORA import. The Pchlide b-dependent import pathway of pPORA thus operates in etiolated seedlings and is switched off during greening. Using dexamethasone-induced RNA interference (RNAi) we tested if PTC52 is involved in controlling both, pPORA import and Pchlide homeostasis in planta. As shown here, RNAi plants deprived of PTC52 transcript and PTC52 protein were unable to import pPORA and died as a result of excess Pchlide a accumulation causing singlet oxygen formation during greening. In genetic studies, no homozygous ptc52 knock-out mutants could be obtained presumably as a result of embryo lethality, suggesting a role for PTC52 in the initial greening of plant embryos. Phylogenetic studies identified PTC52-like genes amongst unicellular photosynthetic bacteria and higher plants, suggesting that the biochemical function associated with PTC52 may have an ancient evolutionary origin. PTC52 also harbors conserved motifs with bacterial oxygenases such as the terminal oxygenase component of 3-ketosteroid 9-alpha-hydroxylase (KshA) from Rhodococcus rhodochrous. 3D-modeling of PTC52 structure permitted the prediction of amino acid residues that contribute to the substrate specificity of this enzyme. In vitro-mutagenesis was used to test the predicted PTC52 model and provide insights into the reaction mechanism of this Rieske non-heme oxygenase.

Phenolic compounds are among the most diverse and widespread of specialized plant compounds and underly many important agronomic traits. Our comprehensive analysis of the maize genome unraveled new aspects of the genes involved in phenylpropanoid, monolignol, and flavonoid production in this important crop. Remarkably, just 19 genes accounted for 70 % of the overall mRNA accumulation of these genes across 95 tissues, indicating that these are the main contributors to the flux of phenolic metabolites. Eighty genes with intermediate to low expression play minor and more specialized roles. Remaining genes are likely undergoing loss of function or are expressed in limited cell types. Phylogenetic and expression analyses revealed which members of gene families governing metabolic entry and branch points exhibit duplication, subfunctionalization, or loss of function. Co-expression analysis applied to genes in sequential biosynthetic steps revealed that certain isoforms are highly co-expressed and are candidates for metabolic complexes that ensure metabolite delivery to correct cellular compartments. Co-expression of biosynthesis genes with transcription factors discovered connections that provided candidate components for regulatory modules governing this pathway. Our study provides a comprehensive analysis of maize phenylpropanoid related genes, identifies major pathway contributors, and novel candidate enzymatic and regulatory modules of the metabolic network.

AbstractThe sections in this article areIntroductionHistone Acetylation and Transcriptional ActivationHistone MethylationInterplay between Histone Acetylation and Methylation in Transcriptional RegulationConclusions

We describe dropdead1-1 (ded1), an EMS-induced recessive lesion mimic mutant of sorghum. It is characterized by the formation of spreading necrotic lesions that share many attributes with those associated with the maize lethal leaf spot1 (lls1) and Arabidopsis accelerated cell death1 (acd1) mutation. We show that as in lls1, ded1 lesions are initiated by wounding and require light for continued propagation, and that loss of chloroplast integrity is responsible for ded1 cell death. Consistent with these parallels, we demonstrate that ded1 is an ortholog of lls1 and encodes pheophorbide a oxidase (PaO) with 93% identity at the protein level. The mutant ded1 allele resulted from a stop codon-inducing single base pair change in exon 6 of the sorghum ortholog of lls1. The ded1 transcript was rapidly and transiently induced after wounding and substantially elevated in leaves containing ded1 lesions. Given that PaO is a key enzyme of the chlorophyll degradation pathway, its dysfunction would result in the accumulation of pheophorbide, a potent photosensitizer that results in the production of singlet oxygen. Consistent with this, cell death associated with ded1 lesions is most likely caused by singlet oxygen as our results exclude superoxide and H2O2 from this role. We explore the signal responsible for the propagation of lesions affecting both ded1 and lls1 lesions and find that both developmental age and ethylene increase the rate of lesion expansion in both mutants.

Chloroplasts and mitochondria contain a family of putative preprotein and amino acid transporters designated PRAT. Here, we analyzed the role of two previously characterized PRAT protein family members, encoded by At3g49560 (HP30) and At5g24650 (HP30-2), in planta using a combination of genetic, cell biological and biochemical approaches. Expression studies and green fluorescent protein tagging identified HP30-2 both in chloroplasts and mitochondria, whereas HP30 was located exclusively in chloroplasts. Biochemical evidence was obtained for an association of mitochondrial HP30-2 with two distinct protein complexes, one containing the inner membrane translocase TIM22 and the other containing an alternative NAD(P)H dehydrogenase subunit (NDC1) implicated in a respiratory complex 1-like electron transport chain. Through its association with TIM22, HP30-2 is involved in the uptake of carrier proteins and other, hydrophobic membrane proteins lacking cleavable NH2 -terminal presequences, whereas HP30-2's interaction with NDC1 may permit controlling mitochondrial biogenesis and activity.

ZmMYB31 and ZmMYB42 are R2R3-MYB transcription factors implicated in the regulation of phenylpropanoid genes in maize. Here, we tested the hypothesis that the regulatory function of MYB31 and MYB42 is conserved in other monocots, specifically in sorghum and rice. We demonstrate that syntelogs of MYB31 and MYB42 do bind to phenylpropanoid genes that function in all stages of the pathway and in different tissues along the developmental gradient of seedling leaves. We found that caffeic acid O-methyltransferase (COMT1) is a common target of MYB31 and MYB42 in the mature leaf tissues of maize, sorghum and rice, as evidenced by Chromatin immunoprecipitation (ChIP) experiments. In contrast, 4-coumarate-CoA ligase (4CL2), ferulate-5-hydroxylase (F5H), and caffeoyl shikimate esterase (CSE), were targeted by MYB31 or MYB42, but in a more species-specific fashion. Our results revealed MYB31 and MYB42 participation in auto- and cross-regulation in all three species. Apart from a limited conservation of regulatory modules, MYB31 and MYB42 syntelogs appear to have undergone subfunctionalization following gene duplication and divergence of maize, sorghum, and rice. Elucidating the different regulatory roles of these syntelogs in the context of positive transcriptional activators may help guide attempts to alter the flux of intermediates towards lignin production in biofuel grasses.

The translation of the genotype into phenotype, represented for example by the expression of genes encoding enzymes required for the biosynthesis of phytochemicals that are important for interaction of plants with the environment, is largely carried out by transcription factors (TFs) that recognize specific cis-regulatory elements in the genes that they control. TFs and their target genes are organized in gene regulatory networks (GRNs), and thus uncovering GRN architecture presents an important biological challenge necessary to explain gene regulation. Linking TFs to the genes they control, central to understanding GRNs, can be carried out using gene- or TF-centered approaches. In this study, we employed a gene-centered approach utilizing the yeast one-hybrid assay to generate a network of protein-DNA interactions that participate in the transcriptional control of genes involved in the biosynthesis of maize phenolic compounds including general phenylpropanoids, lignins, and flavonoids. We identified 1100 protein-DNA interactions involving 54 phenolic gene promoters and 568 TFs. A set of 11 TFs recognized 10 or more promoters, suggesting a role in coordinating pathway gene expression. The integration of the gene-centered network with information derived from TF-centered approaches provides a foundation for a phenolics GRN characterized by interlaced feed-forward loops that link developmental regulators with biosynthetic genes.

This chapter examines the origins, methodological properties, and evolution of school effectiveness research (SER), and the broader and more recent focus of educational effectiveness research (EER), over the last half century. It provides a brief overview of the main disciplinary roots and examines the essential features that underpin the two fields. It also seeks to identify some of the key features of more recent work, conducted in the twenty-first century, showing how the field has evolved since the publication of the first international handbook in 2000 (Teddlie and Reynolds, 2000).

In this article, we address three broad conjectures about what really matters with respect to school effectiveness. Our review of previous evidence prompted us to look at three sets of factors connected with classroom teachers, school policies and processes, and matters of governance. All three have featured prominently in the public arena. In particular, we look for the relative contributions of teacher-, school-, and governance indicators for educational effectiveness (measured by Math achievement). About 100 Dutch primary schools form the database together with findings of international school effectiveness research (studies, reviews, and meta-analyses). School-level variables are the most substantial in explaining educational effectiveness. The sector effect (public/private) explains 16% of the between school variance, other school-level variables explain 51%, and the teacher-or classroom-level variables explain 32%. Some of the underlying variables are identified and we address three broad conjectures about what really matters with respect to school effectiveness.

Tetrapyrroles such as chlorophyll, heme, and bacteriochlorophyll play fundamental roles in the energy absorption and transduction of all photosynthetic organisms. They are synthesized via a complex pathway taking place in chloroplasts. Chlorophyll biosynthesis in angiosperms involves 16 steps of which only one is light-requiring and driven by the NADPH:protochlorophyllide oxidoreductase (POR). Three POR isoforms have been identified in Arabidopsis thaliana--designated PORA, PORB, and PORC--that are differentially expressed in etiolated, light-exposed, and light-adapted plants. All three isoforms are encoded by nuclear genes, are synthesized as larger precursors in the cytosol (pPORs), and are imported posttranslationally into the plastid compartment. Import of the precursor to the dark-specific isoform PORA (pPORA) is protochlorophyllide (Pchlide)-dependent and due to the operation of a unique translocon complex dubbed PTC (Pchlide-dependent translocon complex) in the plastid envelope. Here, we identified a ∼30-kDa protein that participates in pPORA import. The ∼30-kDa protein is identical to the previously identified CELL GROWTH DEFECT FACTOR 1 (CDF1) in Arabidopsis that is conserved in higher plants and Synechocystis. CDF1 operates in pPORA import and stabilization and hereby acts as a chaperone for PORA protein translocation. CDF1 permits tight interactions between Pchlide synthesized in the plastid envelope and the importing PORA polypeptide chain such that no photoexcitative damage occurs through the generation of singlet oxygen operating as a cell death inducer. Together, our results identify an ancient mechanism dating back to the endosymbiotic origin of chloroplasts as a key element of Pchlide-dependent pPORA import.

Core promoters are crucial for gene regulation, providing blueprints for the assembly of transcriptional machinery at transcription start sites (TSSs). Empirically, TSSs define the coordinates of core promoters and other regulatory sequences. Thus, experimental TSS identification provides an essential step in the characterization of promoters and their features. Here, we describe the application of CAGE (cap analysis of gene expression) to identify genome-wide TSSs used in root and shoot tissues of two maize (Zea mays) inbred lines (B73 and Mo17). Our studies indicate that most TSS clusters are sharp in maize, similar to mice, but distinct from Arabidopsis thaliana, Drosophila melanogaster, or zebra fish, in which a majority of genes have broad-shaped TSS clusters. We established that ∼38% of maize promoters are characterized by a broader TATA-motif consensus, and this motif is significantly enriched in genes with sharp TSSs. A noteworthy plasticity in TSS usage between tissues and inbreds was uncovered, with ∼1500 genes showing significantly different dominant TSSs, sometimes affecting protein sequence by providing alternate translation initiation codons. We experimentally characterized instances in which this differential TSS utilization results in protein isoforms with additional domains or targeted to distinct subcellular compartments. These results provide important insights into TSS selection and gene expression in an agronomically important crop.